Neostriatal cholinergic (ChAT) interneurons play an important role in the selection and acquisition of adaptive behavioral actions by encoding the salience and reinforcement value of external events. This information is represented in the precise temporal structure of synchronous multiphasic population responses given to the presentation of behaviorally significant stimuli. An important problem is to understand how these transient signals of ChAT interneurons are detected and decoded in the neostriatal network. Our hypothesis, formulated on the basis of preliminary experiments, is that ChAT interneurons control the activity of the striatal circuitry using nicotinic receptor mediated mechanisms to activate multiple parallel GABAergic mechanisms that elicit kinetically distinct independent inhibitory responses in the spiny projection neurons (SPNs) and in other striatal neurons. These responses originate in part from direct activation of multiple as yet unidentified types of GABAergic interneurons that are distinct from the parvalbumin (PV) containing fast spiking (FS) and NPY expressing interneurons, possibly also involve presynaptic nicotinic facilitation of GABA release from axon terminals and together provide selective, cell type specific inputs to SPNs, ChAT and other interneurons. Using in vitro optogenetic inhibition we also reproduced the population response of ChAT interneurons most commonly observed in behaving animals consisting of a pause of firing followed by weakly synchronous rebound-excitation and demonstrated that this behaviorally directly relevant pattern of activity also engages the inhibitory mechanisms described above. Since these responses are sufficient to inhibit action potential generation in large populations of SPNs they are likely to exert significant effects on the functioning of the basal ganglia and regulate ongoing behavior. The proposed experiments aim at understanding the functional organization of these GABAergic circuits and mechanisms and to quantitatively characterize the effects of reinforcement related population responses of ChAT interneurons on the activity of SPNs in vitro and in vivo. Optogenetic methods will be used to control the activity of ChAT and other interneurons in double transgenic animals allowing the genetic targeting and identification of specific cell types. The disynaptic circuits of ChAT interneurons will be analyzed by identifying the GABAergic interneurons activated by ChAT interneurons and then determining the contribution of individual presynaptic cell types to the various GABAergic response components in SPNs and other neurons by selective optogenetic activation of populations of specific classes of interneurons and by examining the effects of optogenetic inhibition of various interneuron types on IPSCs elicited disynaptically by ChAT interneurons. Finally, we will quantitatively characterize the effects of optogenetically reproduced pause-excitation responses of ChAT interneurons on SPNs in vivo and in vitro. These experiments will describe the functional organization of a powerful novel circuit mechanism of the neostriatum and therefore will significantly advance the understanding of the functioning of the basal ganglia. PUBLIC HEALTH RELEVANCE: Numerous important neurological and psychiatric disorders including Parkinson's and Huntington's diseases, schizophrenia, Tourette's syndrome, obsessive compulsive disorder arise from pathological conditions of the neostriatum. Several of these are directly or indirectly linked to a major neuromodultory system the cholinergic interneurons of this structure, yet critically important issues about the functional role of these interneurons remains unanswered. The proposal will elucidate the organization and functional role of a newly discovered circuit mechanism that is responsible for mediating the reinforcement related population responses of these neurons which under pathological conditions may directly contribute to specific behavioral abnormalities associated with these disorders.